Mouse models of autism

U.S. researchers have engineered mouse
strains that model three relatively common genetic causes of autism spectrum
disorder.1-3 The mice could help researchers figure out the
similarities and differences between various forms of ASD and develop therapeutics
tailored for specific subsets of ASD patients. Understanding the precise
contribution of the three ASD-linked genetic alterations to the specific
clinical manifestations of ASD will require the development of standardized
behavioral and neurophysiological assays for the mutant mice.

The majority of ASD cases have no definitive cause. Only
20%-25% of cases are associated with genetic alterations, most of which are
subtle mutations or changes in gene copy number of individual genes or small
chromosomal regions. A handful of genetically-based ASD cases result from
large-scale chromosomal abnormalities.

"Dozens of causes of autism have now been identified"
through genomic methods, said Randall Carpenter. The challenge, he said, is to
unravel how these genetic alterations lead to disease and to determine the
relevance of these disease mechanisms for the majority of patients without
genetic abnormalities.

Carpenter is cofounder, president and CEO of Seaside Therapeutics Inc.,
which is developing therapies for Fragile X syndrome and ASD.

The findings also reveal how ASD-related genes affect
neurons and other brain cells involved in g-aminobutyric acid
(GABA)
signaling and glutamate signaling.

Locus pocus

Two of the new mouse
strains were designed to test the effects of deletions or duplications in a
pair of chromosomal regions previously associated with ASD in human genomic
studies.

A team led by Alea Mills, professor of genetics at Cold
Spring Harbor, tackled the 16p11.2 chromosomal locus, which she said is deleted
in "about 1% of autistic children, making it one of the strongest genetic
factors" in ASD. The region contains 27 genes with a variety of functions,
and it is unknown which contribute to ASD.1

The team made mice with one or three copies of the 16p11.2
locus, thus altering the levels of proteins encoded by the region.

She said her team "generated mice with altered dosage
of the same 27 genes as patients with ASD, without any preconceived notions of
what the molecular mechanisms might be."

Mice with only a single copy of the 16p11.2 locus had a
high rate of neonatal mortality, and those that survived to adulthood had
behavior that was reminiscent of ASD compared with wild-type littermates.

"In the heterozygous deletion, we saw sleep disorders,
hyperactivity and highly repetitive behavior when we challenged these animals
with a new environment," said Mills. "We also saw that eight regions
of the brain are larger in the deletion mouse than in controls. This could be
analogous to the increased head circumference measurements in ASD patients"
compared with in individuals without ASD.

The team also made mice with an extra copy of the locus,
but that strain behaved similarly to wild-type animals.

Results were published in the Proceedings of the National Academy of Sciences.

Mills now plans to drill down into the 16p11.2 region to
find the specific genes responsible for the heterozygous knockout mouse's
behavioral and neurophysiological phenotypes.

She has filed for a patent covering the 16p11.2 transgenic
and heterozygous knockout mice.

One gene at a time

A Harvard Medical School
team led by Matthew Anderson took a more targeted tack than Mills and focused
on a single gene-ubiquitin protein ligase E3A (UBE3A; E6AP)-in a genomic region associated
with another form of hereditary ASD.2

UBE3A
is part of the 15q11-13 region, which occurs as a duplicate or triplicate in 1%-3%
of patients with ASD.

Anderson hypothesized that this genetic alteration would be
a good starting point for building ASD models. He said patients carrying extra
copies of the 15q11-13 locus have a relatively straightforward form of ASD
without the complex pathophysiological features of larger chromosomal
alterations that cause the severe, syndromic forms of ASD.

"This is the most common genetic change in
nonsyndromic autism patients," said Anderson.

Previous mouse studies by other researchers established
that duplicating the entire region led to abnormal behavior.4
Anderson therefore hypothesized that increasing the number of copies of the Ube3a gene alone could result in an
ASD-like condition in mice.

Compared with wild-type controls, mice with an extra copy
of Ube3a had impaired
social behavior, decreased vocalization in response to mouse social cues and
increased repetitive self-grooming.

Anderson thinks these behavioral abnormalities are
potentially indicative of ASD-like pathology and could thus be good assays for
therapeutics. His next step is to compare the neurological characteristics of
mice with extra copies of Ube3a
against wild-type controls and Ube3a
knockouts to understand what brain circuits are most affected by the gene.

Results were published in Science Translational Medicine. Anderson has filed
patents on the discoveries, and the IP is available for licensing.

A rare loss-of-function mutation in that gene causes a
syndrome of epilepsy, intellectual disability and autism. In 2008, genomewide
association studies linked variations in CNTNAP2
to increased risk of ASD.5-7

Researchers led by Daniel Geschwind, professor of
neurology, psychiatry and human genetics at UCLA, found that Cntnap2 knockout mice had
behavioral problems including hyperactivity, repetitive behavior and awkward
social interactions compared with wild-type controls. The knockout mice also
were prone to seizures and had an abnormal electroencephalogram pattern.

Results were published in Cell and were not subject to patents.

Model behavior

One question going
forward is whether studying the behavior of these three mouse models will be
useful for understanding the disease process in the majority of ASD cases that
lack such genetic alterations.

Robert Ring, VP of translation research at patient
advocacy group Autism Speaks, cautioned that even
though these studies present faithful models of the genetic lesions underlying
some rare forms of ASD, the behavioral assays used by the researchers have yet
to be validated.

"I'm not sure that we know how informative these
mouse assays are," said Ring. "We can take highly penetrant genetic
risk factors and make animal models, but how much these actually replicate the
etiology of the neurodevelopmental problem in autism is unclear."

To resolve the question of how to interpret mouse
behavior, Ring said Autism Speaks hopes to launch an effort to standardize
phenotypic assays for mouse models.

"We have been thinking about funding a network of
standardized assays to ensure there's consistency in the data," said Ring.

Geschwind, however, thinks the three new models could be
relevant to a wide range of ASD cases. He said despite the distinct molecular
causes, there was a surprising amount of similarity in the behavioral
abnormalities of the three animal models. In addition, he said, those
behavioral abnormalities resemble ASD in humans.

"Considering that there is no single mutation that
causes more than 0.5% of cases of ASD, it's remarkable that in multiple models
you can have genes which to some extent parallel what goes on in patients with
ASD," said Geschwind.

Thus, the mice might point to common pathophysiological
features among many ASD patients that could be counteracted with pharmacological
intervention.

Ring said distinct neurological abnormalities can lead to
symptoms that are similar from a behavioral standpoint but nonetheless may
require different therapeutic interventions.

Neuron the right track

For example, Geschwind's
team found that Cntnap2
knockouts had lower numbers of inhibitory GABAergic neurons than wild-type
controls. This suggests agonizing the inhibitory GABAergic circuit could have
beneficial effects.

Likewise, Anderson's team found evidence of decreased
glutamatergic signaling in mice bearing extra copies of Ube3a.

"One of the exciting ideas in ASD is an imbalance of
inhibitory and excitatory circuits," said Geschwind. It may be possible to
correct this imbalance by treating with agonists or antagonists of whatever
circuit is defective in any given mouse model, he noted.

As additional mouse models of ASD come online, Anderson
said it may be possible to categorize the animals according to the nature of
their signaling problems. Doing so could help identify subsets of ASD patients
likely to respond to a given drug.

Indeed, Ring thinks the signaling alterations in the new
mouse models might be better guideposts for therapeutic development than the
behavioral phenotypes.

For now, drug developers will need more detailed
information about the molecular defects caused by various ASD-associated
chromosomal lesions before deciding how to interpret the mouse model data.

"We prioritize our research around single genes that
we know are mutated and cause severe disorders," said Carpenter. "As
you get into more complex situations like copy number variants, it gets harder
to interpret the effects of these lesions on mouse behavior."

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